The present invention relates generally to the detection of human herpes viruses and, more particularly, to measuring human herpes viral infection in real time, for example, for purposes of clinical testing, drug screening and infection prognostication, inter alia.
Eight human herpes viruses are recognized presently, and these are divided into three sub-families, as shown below.
1.Alpha herpes virinaeSimplex virushuman herpes virus 1 (HHV1);human herpes virus 2 (HHV2)Varicellovirushuman herpes virus 3 (HHV3)2.Beta herpes virinaeCytomegalovirushuman herpes virus 5 (HHV5)Roseolovirushuman herpes virus 6 (HHV6);human herpes virus 7 (HHV7)3.Gamma herpes virinaeLymphocryptovirushuman herpes virus 4 (HHV4)Rhadinovirushuman herpes virus 8 (HHV8)
Various of these viruses are associated with human pathologies. For instance, HHV4 or “Epstein-Barr virus” (EBV) is associated with infectious mononucleosis, also known as “glandular fever.” HHV4 is linked as well with oncogenesis with regard, for example, in Burkitt's lymphoma and nasopharyngeocarcinoma. Additionally, HHV4 is found in immune-suppressed patients and in patients suffering from Hodgkin's disease.
HHV5 is known as “cytomegalovirus” and causes infections in the lungs of immune-suppressed persons. HHV5 infection is less common than HHV4, and yet approximately 80% of the population in the United Kingdom, for instance, experience HHV5 infection by mid-life. HHV5 is probably transmitted by means of saliva, sexual contact, droplets and blood transfusions. In addition, both HHV4 and HHV5 are believed to be associated with chronic-fatigue syndrome, a malady that may afflict as many as in six out of every 100,000 people.
HHV6 is associated with “roseola” and “infantum” infections in children and with immune-compromised patients. For example, AIDS patients exhibit HHV6 infection, although the significance of the HHV6 infection is unclear. HHV6 is susceptible to antiviral drugs. It is unclear, however, how antiviral drugs work against HHV6 or how resistance to such drugs develops. A significant aspect of HHV6 infection is its putative tie-in with multiple sclerosis (MS) and chronic fatigue syndrome (CFS), respectively.
Less is known about HHV7 and HHV8. No clear evidence for the direct involvement of HHV7 in any human disease has been reported. Studies indicate, however, that HHV7 may be associated with HHV6-related infections. In a related vein, HHV8 infection is believed to be associated with Karposi's Sarcoma.
Conventional techniques have several limitations that have retarded their use in elucidating the significance of human herpes virus infection in the development of several diseases, such as CFS. Prevailing methods are not sensitive for detection of specific types and strains of human herpes virus and for quantitation of specific human herpes viruses. If an assay reveals a positive viral load, therefore, additional assays are required to detect specific human herpes viruses. As a consequence, several days may elapse before a patient is diagnosed. In addition, conventional techniques do not detect virus when viral levels are low, for example, at early stages of infection. Accordingly, clinical settings are hampered by a lack of sensitive methods to detect virus and lose valuable time before a patient can receive treatment.
Current methods also require large amounts of physiological samples, whereas only small amounts of samples from each patient are usually available for analysis. It is therefore difficult to generate reproducible measurements of viral levels from peripheral blood of patients. Additionally, small amounts of physiological samples may be difficult to obtain and can entail procedures risky to patients, as in the instance of cerebrospinal fluid, and therefore may not have not been available for routine testing in a clinical environment.
Conventional approaches in this area often involve the polymerase chain reaction (PCR), by which smaller amount of samples can be analyzed. PCR-based testing for human herpes virus has been limited, however, by long turnaround times. Moreover, the application of PCR to small samples can lead to an increase in error rates, because more amplification cycles are required for lesser amounts of sample. In addition, PCR-based results for human herpes viruses have appeared to lack reproducibility, given the conflicting date that are reported in the literature.
The absence of a treatment for many human herpes viruses is another important concern. There is no practical approach to monitoring the effectiveness of therapeutic agents in the clinical setting, reflecting the difficulties in testing for specific virus infections and, particularly, in quantitating viral levels. Furthermore, the lack of a suitable screening approach for such therapeutic agents detracts from the incentive to develop them.
These difficulties pertaining to conventional PCR-based approaches to diagnosis are illustrated by attempts to measure HHV6 infection. Reports on the use of PCR to measure HHV6 DNA in CFS and MS patients indicate that the viral levels thus detected depend upon the particular methodology employed. For example, Locatelli et al., WO 00/29613, used real-time quantitative PCR to obtain HHV 6-positive results in 34% of cerebral spinal fluid (CSF) and 18% of plasma samples from MS patients. Similarly, Soldan et al., Nature Med. 3: 1394-1397 (1997), used a nested PCR procedure and found that a significant number of plasma samples from MS patients were HHV6-positive. On the other hand, other labs also employing PCR-based assays reported much lower levels of HHV 6 DNA in MS patients. For example, Ablashi et al., J. Clinic. Virol. 16(3): 179-191 (2000), reported that 9.1% of CSF and 4.5% of plasma samples tested positive for HHV 6, whereas Taus et al., Acta. Neurol. Scand. 101(4): 224-228 (2000), reported that HHV6 DNA was reported absent from CSF drawn from MS patients.
Similarly, other groups relying on PCR-based methods have reported low amounts of HHV6 in CFS patients. Secchiero et al., J. Infect. Dis. 171: 273-280 (2000), for example, reported that 2.6% of plasma samples from CFS patients were HHV6-positive.
In summary, prevailing PCR methods for human herpes virus yield irreproducible results, which complicate efforts to diagnose and treat HHV infection. Further, these methods have not addressed the need for a fast throughput assay, which is essential in clinical settings. As noted, moreover, there is a lack of therapeutic measures against many human herpes viruses, such as HHV6a and HHV6b, in part due to the absence of a ready technique for detecting them.